This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Our program correlates time-resolved X-ray diffraction of actively working muscle with 3-D reconstructions from muscles quick-frozen/freeze-substituted for electron microscopy. We aim to characterize the structure and dynamic choreography between motor proteins (myosin on actin) and the regulatory proteins (troponin and tropomyosin) in striated muscle. Mechanical triggering by stretch is important to the physiological properties (Frank-Starling behavior) of human cardiac muscle, and is essential to flight in most insects (>half of all known animal species!), but the underlying mechanism has been unknown. Recently published 3D reconstructions from our lab identified a newly recognized class of connections between myosin and troponin ('troponin bridges'), and suggested a molecular mechanism which we have tested with real-time X-ray diffraction 'movies'of contracting insect flight muscle that were sinusoidally stretch-activated. Results were consistent with the hypothesis that troponin bridges mechanically tug tropomyosin aside during stretch activation, exposing myosin-binding sites on actin and allowing subsequent delayed force generation, but only in the presence of calcium. In the absence of calcium, stretching relaxed muscles caused observable movements of the troponin bridges but only restricted movement of tropomyosin. The X-ray movies also revealed an unexpected twisting of the helical myosin filaments, a feature which may be unique to insect flight muscle. Current experiments are aimed at dissecting the interplay between stretch and calcium, and identifying more specifically the exact chemical partners that make up troponin bridges, with an eye towards exploring how analogous mechanical linkages might operate in other muscles, such as mammalian cardiac muscle.